We will use cloned genomic fragments from the 1,000-kilobase region of DNA amplified in PALA-resistant hamster cells to investigate the details of the amplification process, especially important structural features of the amplified unit, whether or not each cloned region is amplified to the same extent in independent events, whether or not transcriptional units other than the CAD gene are present within the amplified region and, if so, whether or not they are expressed in proportion to gene copy number. We also plan to look at cells early in the process of gene amplification in an attempt to characterize early structures and events in the process. The general method used to clone amplified genomic fragments is being extended to cDNA libraries, which will be probed with genomic DNAs. Differential hybridization of a cDNA clone signifies that the corresponding mRNA is represented more often in one genomic DNA probe than in the other. The method will be used to look generally for gene amplification in association with several types of drug resistance, including multiple drug cross-resistance, and in association with transformation (tumorigenesis).